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ObjectiveTo describe the clinical, histopathological and molecular features of a novel inherited SRY allele (p.Met64Val; consensus box position 9) observed within an extensive pedigree: two 46, XY sisters with primary amenorrhea (16 and 14 years of age; probands P1 and P2), their normal father and brother, and an affected paternal XY grandaunt.Design, Setting, Participants and Outcome MeasurementsFollowing DNA sequencing to identify the SRY mutation, hormonal studies of the probands and histopathological examination of their gonads were performed. Functional consequences of p.Met64Val (and other mutations at this site) were also investigated.ResultsBreast development in P1 and P2 was Tanner II and IV, respectively. Müllerian structures and gonads resembling ovaries were found in each sister. Histopathology revealed gonadal dysgenesis, gonadablastoma and dysgerminoma. AMH/MIS, P450 SCC, and P450 aromatase were expressed in gonadoblastoma tissues. Genomic sequencing revealed no candidate mutations in other genes related to sexual differentiation. Variant p.Met64Val impaired Sox9 transcriptional activation associated with attenuated occupancy of the testis-specific enhancers Enh13 and TESCO. Biophysical studies indicated that the mutant HMG box retains specific DNA binding and DNA bending but with accelerated rate of protein-DNA dissociation.ConclusionThe partial biological activity of p.Met64Val SRY, maintained at the threshold of SRY function, rationalizes opposing paternal and proband phenotypes (the “the father-daughter paradox”). Steroidal biosynthesis by gonadoblastoma may delay genetic diagnosis and recognition of gonadal tumors. Quantitative assessment of inherited SRY alleles highlights the tenuous transcriptional threshold of developmental decision-making in the bipotential gonadal ridge.

Context: SRY, an architectural transcription factor containing a SOX-related high-mobility group (HMG) box, initiates testis formation in the mammalian bipotential gonadal ridge. Inherited human SRY mutations can be associated with differences in sexual differentiation (DSD) with variable phenotypes in a family. Objective: To describe the clinical, histopathological and molecular features of a novel inherited SRY allele (pMet64Val; consensus box position 9) observed within an extensive pedigree: two 46, XY sisters with primary amenorrhea (16 and 14 years of age; probands P1 and P2), their normal father and brother, and an affected paternal XY grandaunt. Design, Setting, Participants and Outcome Measurements: Following DNA sequencing to identify the SRY mutation, hormonal studies of the probands and histopathological examination of their gonads were performed. Functional consequences of p.Met64Val (and other mutations at this site) were also investigated. Results: Breast development in P1 and P2was Tanner II and IV, respectively. Müllerian structures and gonads resembling ovaries were found in each sister. Histopathology revealed gonadal dysgenesis, gonadoblastoma and dysgerminoma. AMH/MIS, P450 SCC, and P450 aromatase were expressed in gonadoblastoma tissues. Variant p.Met64Val impaired Sox9 transcriptional activation associated with attenuated occupancy of the testis-specific enhancersEnh13 and TESCO. Conclusion: The partial biological activity of p.Met64Val SRY, maintained at the threshold of SRY function, rationalizes opposing paternal and proband phenotypes (the “the father-daughter paradox”).Sex steroids biosynthesis by gonadoblastoma may delay genetic diagnosis and recognition of gonadal tumors. Quantitative assessment of inherited SRY alleles highlights the tenuous transcriptional threshold of developmental decision-making in the bipotential gonadal ridge.

The human adrenal cortex, involved in adaptive responses to stress, fluid homeostasis, and secondary sexual characteristics, arises from a tightly regulated development of a zone and cell type-specific secretory pattern. However, the molecular mechanisms governing adrenal zonation, particularly postnatal ZR development, which produces adrenal androgens in a life-time-specific manner, remain poorly understood. The hallmark of ZR is the low expression of type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). However, the mechanisms underlying HSD3B2 downregulation in the ZR remain unknown. MiRNAs are seen as regulators of cell phenotypes. The objective of the study was to compare miRNA expression profiles in human adrenal ZR and zona fasciculata (ZF). ZF and ZR were microdissected from human adrenals tissues (n=5, from boys aged 16, 17 and 19 yr and girls aged 12 and 16 yr) by laser capture microdissection (LCM). Total RNA was extracted from 5 ZF/ZR pairs and next-generation sequencing (NGS) was used to perform the microRNA expression profiling. 281 mature microRNAs were identified in human adrenal cortex. Among them, 7 microRNAs were significantly differentially expressed between ZF and ZR. The expression of miR-375-3p, miR-483-3p and miR-7-5p was higher in ZR compared with its paired ZF by 2-fold or greater. Multiple available bioinformatic algorithms (TargetScan, miRanda, DianaLab and PicTar) were employed to search for its target genes. Among predicted target genes, several genes (GATA-6, GATA-4, SF1, NR4A2, and IGF-1) are known to be involved in HSD3B2 regulation. In summary, LCM combined with NGS provided a robust approach to explore the adrenal zone-specific micro-RNA profiling. Our data gave first hints that miRNAs might be novel regulatory modules associated with human adrenal ZR cell-specific transcript regulation underlying developmental androgen production.

The human adrenal cortex, involved in adaptive responses to stress, fluid homeostasis, and secondary sexual characteristics, arises from a tightly regulated development of a zone and cell type-specific secretory pattern. However, the molecular mechanisms governing adrenal zonation, particularly postnatal zona reticularis development, which produce adrenal androgens in a lifetime-specific manner, remain poorly understood. Epigenetic events, including DNA and histone modifications as well as regulation by noncoding RNAs, are crucial in establishing or maintaining the expression pattern of specific genes and thus contribute to the stability of a specific differentiation state. Emerging evidence points to epigenetics as another regulatory layer that could contribute to establishing the adrenal zone-specific pattern of enzyme expression. Here, we outline the developmental milestones of the human adrenal cortex, focusing on current advances and understanding of epigenetic regulation of postnatal functional zonation. Numerous questions remain to be addressed emphasizing the need for additional investigations to elucidate the role of epigenetics in the human adrenal gland. Ultimately, improved understanding of the epigenetic factors involved in adrenal development and function could lead to novel therapeutic interventions.

We hypothesized that DNA methylation is involved in human adrenal functional zonation. mRNAsexpression and methylation pattern of RARB, NR4A1 and HSD3B2 genes in human adrenal tissues (HAT)and in pediatric virilizing adrenocortical tumors (VAT) were analyzed. For analysis of the results sampleswere divided into 3 age groups according to FeZ involution, pre and post-adrenarche ages. In all HAT,similar RARB mRNA was found including microdissected zona reticularis (ZR) and zona fasciculata, butHSD3B2 and NR4A1 mRNAs were lower in ZR (p < 0.05). NR4A1 and RARB promoters remainedunmethylated in HAT and VAT. No adrenal zone-specific differences in NR4A1 methylation wereobserved.In summary, RARB was not associated with ZR-specific downregulation of HSD3B2 in postnatal humanadrenocotical zonation. DNA methylation would not be involved in NR4A1 adrenocortical cell-typespecific downregulation. Lack of CpG islands in HSD3B2 suggested that HSD3B2 ZR-specific downregulationwould not be directly mediated by DNA methylation. Fil: Baquedano, María Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Perez Garrido, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Goñi, Javier. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Saraco, Nora Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Aliberti, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Berensztein, Esperanza Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rivarola, Marco Aurelio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Belgorosky, Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina

Estrogens play important role in the placenta throughout the gestation. Human placenta (PL) expresses high levels of ERRγ type 1 isoform as well as Aromatase (Aro).ERRγ1has also be described as possibly implicated in the regulation of theenergy metabolism in PL, however its physiological role is not yet clear.Aro is the key enzyme for estrogen biosynthesis from androgens and in human placenta is expressed exclusively in syncytiotrophoblast. It has been described that ERRγ serves as an oxygen-responsive transcription factor that regulates Aro gene expression in PL.We have previously described splicing variants of AromRNA(1-2). Our aim was to analyze ERRγ1 mRNA expression and Aro mRNA active variant expression in PL from term LGA and preterm (PT) (

3βHSD2 enzyme is crucial for adrenal and gonad steroid biosynthesis. In enzyme deficiency states, due to recessive loss-of-function HSD3B2 mutations, steroid flux is altered and clinical manifestations result. Deficiency of 3βHSD2 activity in the adrenals precludes normal aldosterone and cortisol synthesis and the alternative backdoor and 11-oxygenated C19 steroid pathways and the flooding of cortisol precursors along the Δ5 pathway with a marked rise in DHEA and DHEAS production. In gonads, it precludes normal T and estrogen synthesis. Here, we review androgen-dependent male differentiation of the external genitalia in humans and link this to female development and steroidogenesis in the developing adrenal cortex. The molecular mechanisms governing postnatal adrenal cortex zonation and ZR development were also revised. This chapter will review relevant clinical, hormonal, and genetic aspects of 3βHSD2 deficiency with emphasis on the significance of alternate fates encountered by steroid hormone precursors in the adrenal gland and gonads. Our current knowledge of the process of steroidogenesis and steroid action is derived from pathological conditions. In humans the 3βHSD2 deficiency represents a model of nature that reinforces our knowledge about the role of the steroidogenic alternative pathway in sex differentiation in both sexes. However, the physiological role of the high serum DHEAS levels in fetal life as well as after adrenarche remains to be elucidated.

Androgen insensitivity syndrome (AIS) is a hereditary condition in patients with a 46,XY karyotype in which loss-of-function mutations of the androgen receptor (AR) gene are responsible for defects in virilization. The aim of this study was to investigate the consequences of the lack of AR activity on germ cell survival and the degree of testicular development reached by these patients by analyzing gonadal tissue from patients with AIS prior to Sertoli cell maturation at puberty. Twenty-three gonads from 13 patients with AIS were assessed and compared to 18 testes from 17 subjects without endocrine disorders. The study of the gonadal structure using conventional microscopy and the ultrastructural characteristics of remnant germ cells using electron microscopy, combined with the immunohistochemical analysis of specific germ cell markers (MAGE-A4 for premeiotic germ cells and of OCT3/4 for gonocytes), enabled us to carry out a thorough investigation of germ cell life in an androgen-insensitive microenvironment throughout prepuberty until young adulthood. Here, we show that germ cell degeneration starts very early, with a marked decrease in number after only 2 years of life, and we demonstrate the permanence of gonocytes in AIS testis samples until puberty, describing 2 different populations. Additionally, our results provide further evidence for the importance of AR signaling in peritubular myoid cells during prepuberty to maintain Sertoli and spermatogonial cell health and survival. Fil: Aliberti, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Perez Garrido, Natalia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Marino, Roxana. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Ramirez, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Solari, Alberto Juan. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Sciurano, Roberta Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Costanzo, Mariana. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Guercio, Gabriela Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Warman, Diana Mónica. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Bailez, Marcela. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Baquedano, María Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Rivarola, Marco Aurelio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Belgorosky, Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Berensztein, Esperanza Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina

Steroid acute regulatory (StAR) protein is a mitochondria-targeted protein that is part of the transduceosome complex crucial for transport of cholesterol to mitochondria. Recessive mutations cause classic and nonclassic congenital lipoid adrenal hyperplasia.The aim of this study was to report the clinical, hormonal, genetic, and functional data of a novel heterozygous mutation in the StAR gene found in a 46,XY patient with ambiguous genitalia and neonatal severe steroidogenic deficiency.Undetectable serum steroids with high ACTH and plasma renin activity but normal acute GnRH response were found in infancy. After gonadectomy (at 3 yr of age), serum LH and testosterone were undetectable, whereas FSH was normal but increased slowly afterward. Estrogen replacement therapy, started at 10.2 yr of age, suppressed gonadotropins (for 2 yr). However, after 1 month off estrogens, the patient showed castrated levels. At 11.9 yr old, after fludrocortisone withdrawal because of hypertension, plasma renin activity and aldosterone remained normal, suggesting mineralocorticoid recovery by a StAR-independent mechanism.We found a de novo heterozygous IVS-2AG StAR mutation and the reported heterozygous p.G146A SF1 polymorphism with normal CYP11A1, FDXR, FDX1, VDAC1, and TSPO genes. The mutant StAR transcript lacked exon 2, resulting in the in-frame loss of amino acids 22 to 59 in the N-terminal mitochondrial targeting signal. In vitro, the mutant protein exhibited reduced StAR activity in a dominant-negative manner and almost no mitochondria localization.A misfolded p.G22_L59del StAR might interfere with wild-type StAR activity by blocking the transduceosome complex, causing an autosomal dominant form of StAR deficiency, explaining the clinical phenotype. We speculated that estrogen might have modulated mineralocorticoid function and pubertal maturation in a human natural model lacking endogenous steroid production.

In humans, steroidogenic factor 1 (NR5A1/SF-1) mutations have been reported to cause gonadal dysgenesis, with or without adrenal failure, in both 46,XY and 46,XX individuals. We have previously reported extreme within-family variability in affected 46,XY patients. Even though low ovarian reserve with preserved fertility has been reported in females harboring NR5A1 gene mutations, fertility has only been observed in one reported case in affected 46,XY individuals. A kindred with multiple affected members presenting gonadal dysgenesis was studied. Four 46,XY individuals presented severe hypospadias at birth, one of them associated with micropenis and cryptorchidism. The other 3 developed spontaneous male puberty, and 1 has fathered 5 children. Four 46,XX patients presented premature ovarian failure (one of them was not available for the study) or high follicle-stimulating hormone levels. Mutational analysis of the NR5A1 gene revealed a novel heterozygous mutation, c.938G→A, predicted to cause a p.Arg313Hys amino acid change. A highly conserved amino acid of the ligand-binding domain of the mature protein is affected, predicting abnormal protein function. We confirm that preserved fertility can be observed in patients with a 46,XY disorder of sex development due to heterozygous mutations in the NR5A1 gene.

Background: Three novel heterozygous SF-1 gene mutations affecting multiple members of two unrelated families with a history of 46,XY disorders of sex development (DSD) and 46,XX ovarian insufficiency are described. Methods: Clinical and mutational analysis of the SF-1 gene in 9 subjects of two families. Results: Family 1 had 2 affected 46,XY DSD subjects. One, born with severe perineal hypospadias, was raised as a male, and presented normal adolescence. The other, born with ambiguous genitalia, uterus, and mild testicular dysgenesis, was raised as a female. A W279X heterozygous mutation and an intronic deletion (g3314-3317delTCTC (IVS 4 + 8) was found in the SF-1 gene. In family 2, 4/6 affected siblings had 46,XY DSD or hypospadias. An affected 46,XX sister had normal sexual development but increased FSH levels. The 37-year-old affected mother had entered menopause. An Y183X heterozygous mutation was detected. Conclusion: An extreme within-family phenotypic variability, ranging from severe prenatal undervirilization to normal pubertal development, was observed in 46,XY-affected siblings, indicating that other unknown factors might be involved in the phenotype. Low ovarian reserve and preserved fertility in 46,XX subjects can be observed in heterozygous SF-1 gene mutations.

Patients with TSH-beta subunit defects and congenital hypothyroidism are missed by TSH-based neonatal screening.Our objective was to report the molecular consequences of a novel splice-junction mutation and a novel missense mutation in the TSH-beta subunit gene found in two patients with congenital central hypothyroidism and conventional treatment-resistant anemia.Patient 1 had a homozygous G to A nucleotide change at the 5' donor splice site of exon/intron 2. This resulted in a silent change at codon 34 of the mature protein. In vitro splicing assays showed that the mutant minigene dramatically affected pre-mRNA processing, causing exon 2 to be completely skipped. The putative product from a new out-of-frame translational start point in exon 3 is expected to yield a nonsense 25-amino-acid peptide. In patient 2, sequence analysis revealed a compound heterozygosis for the already reported 313delT (C105Vfs114X) mutation and for a second novel mutation in exon 3, substituting G for A at cDNA nucleotide position 323, resulting in a C88Y change. This cysteine residue is conserved among all dimeric pituitary and placental glycoprotein hormone-beta subunits. Data from in silico analysis confirmed that the C88Y mutation would affect subunit conformation. Indeed, two different bioinformatics approaches, PolyPhen and SIFT analysis, predicted C88Y to be a damaging substitution.In isolated TSH deficiency, the exact molecular diagnosis is mandatory for diagnosis of isolated pituitary deficiency, delineation of prognosis, and genetic counseling. Moreover, diagnosis of central hypothyroidism should be considered in the face of severe infant anemia of uncertain etiology.

Adrenarche is a process of postnatal sexual maturation occurring in higher primates, in which there is an increase in the secretion of adrenal androgens. It is the consequence of a process of postnatal organogenesis characterized by the development of a new zone in the adrenal cortex, the zona reticularis (ZR). The mechanism of this phenomenon remains poorly understood, suggesting that it might be a multifactorial event. A relationship between circulating IGF-I, insulin sensitivity, and adrenal androgens has been postulated. Boys and girls have different patterns of changes in insulin sensitivity at puberty, perhaps secondary to differences in the estrogen milieu. Estrogen effects may also play a role in premature adrenarche. Peripheral or local IGF-1 actions could regulate adrenal progenitor cell proliferation and migration. Since adrenal progenitor cells as well as IGF-I and the IGF-R1 are located in the outer zone of the adrenal cortex during childhood and adolescence, this peripheral cell layer, below the capsule, may contain undifferentiated progenitor cells. Therefore, the IGF-R1 signaling pathway might positively modulate the proliferation and migration of adrenal progenitor cell to stimulate the development of adrenal zones, including ZR. However, no evidence of a direct action of IGF-I on ZR was found. In addition, a role for estrogens in the ontogenesis of ZR is suggested by the presence of aromatase (CYP19) in the subcapsular zona glomerulosa and in the adrenal medulla. Estrogens produced locally could act on ZR by interacting with estrogen receptor beta (ERbeta), but not alpha, and membrane estrogen receptor GPR-30. An estradiol-induced increase in DHEA/cortisol ratio was indeed seen in cultures of adrenocortical cells from post-adrenarche adrenals. In summary, several lines of evidence point to the action of multiple factors, such as local adrenal maturational changes and peripheral metabolic signals, on postnatal human adrenal gland ZR formation.

Immunoexpression of IGF-I, IGF-II, type 1 IGF receptor (IGFR), insulin receptor (IR), and GH receptor (GHR) was analyzed in human testis, in three age groups (Gr): Gr1 (neonates), Gr2 (postnatal testicular activation), and Gr3 (early prepuberty). In interstitial cells, low IGF-I and GHR, but moderate IR immunoexpression was observed in all Grs. However, high expression of IGF-II in Gr1, and moderate expression of IGFR in Gr1 and Gr2 were found. In Leydig cell (LC), high expression of IGF-II, moderate expression of IGFR and GHR, and undetectable IGF-I was found. Moreover, IR was highly expressed in Gr2. The effect of IGF-I on cell proliferation (PI) and apoptosis (AI), induction of cytochrome P450 side chain cleavage (cP450scc) immunoexpression, 3beta-hydroxysteroid dehydrogenase mRNA and testosterone (T) secretion was evaluated in human testis cell cultures. IGF-I increased P450scc immunoexpression, 3beta-hydroxysteroid dehydrogenase mRNA, T secretion, and PI, but decreased AI. We propose that IGF-II, mainly through IR, is involved in functional LC differentiation. In some interstitial cells, probably in LC precursors, IGF-II/IR could be involved, among other factors, in the stimulation of PI and/or inhibition of AI, and in LC differentiation.

La enzima P450 aromatasa (P450Aro) participa en la síntesis de estrógenos a partir de andrógenos. La mutación c655G>A, descripta en forma heterocigota en una niña y en forma homocigota en un hombre adulto, ambos con déficit de aromatasa, genera la disrupción del sitio dador de splicing exón5-intrón5. Se ha postulado que la retención del intrón5 y la generación de una proteína truncada inactiva serían las consecuencias de esta mutación. Sorpresivamente, la paciente presentó desarrollo espontáneo de mamas y niveles puberales de estradiol, sugiriendo una actividad aromatasa (AA) residual. En principio postulamos que la mutación c655G>A generaría la pérdida del exón5 con conservación del marco de lectura, generándose una proteína con menor actividad que podría explicar el déficit parcial. La expresión del ARNm sin exón5 (ARNm- E5) en linfocitos de la paciente sugiere una asociación entre la pérdida del exón y la presencia de la mutación; posteriormente confirmada realizando ensayos de splicing en células Y1. Sin embargo, la expresión del cDNAE5 en células Y1 presentó una AA nula que no explicaría un déficit parcial. La expresión del ARNm-E5 fue detectada en placenta, testículo y adrenal humanos como una variante de splicing normal. Estos resultados indicarían la ocurrencia de splicing alternativo (SA) en la zona codificante de P450Aro como un posible mecanismo regulador de la producción de estrógenos en tejidos esteroidogénicos humanos. La mutación c655G>A podría alterar los mecanismos fisiológicos reguladores del SA del exón5 favoreciendo su exclusión. De esta forma, bajos niveles de ARNm+E5 podrían expresarse aun en presencia de la mutación explicando el fenotipo de déficit parcial observado en la paciente.P450 aromatase (P450Aro), involved in androgen to estrogen conversion, is encoded by the CYP19 gene. P450Aro c655G>A mutation described in heterozygous form in a girl and in homozygous form in an adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2) levels suggesting residual P450Aro activity (AA). Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patient's lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA) was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patient's phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS) events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655G>A allele explaining residual AA evidenced in the affected girl.

Context: The mechanisms of postnatal adrenal zonation remain unclear.Objective: To provide a clue for a possible role of estrogens in adrenarche, we studied the expression of estrogen receptor (ER)α, ERβ, G protein-coupled receptor (GPR)30, and cP450aromatase (cP450arom) in human adrenal tissue.Design: Human adrenal tissue was collected from three postnatal age groups (Grs): Gr 1, younger than 3 months (n = 12), fetal zone involution; Gr 2, 3 months to 6 yr (n = 17), pre-adrenarche; and Gr 3, older than 6–20 yr (n = 12), post-adrenarche period.Results: ERβ mRNA in Grs 1 and 3 was higher than in Gr 2 (P < 0.05). By immunohistochemistry and laser capture microdissection followed by RT-PCR, ERβ was expressed in zona reticularis and fetal zone, GPR30 in zona glomerulosa (ZG) and adrenal medulla, while ERα mRNA and protein were undetectable. cP450arom mRNA in Gr 3 was higher than in Grs 1 and 2 (P < 0.05), and localized to ZG and adrenal medulla by laser capture microdissection. cP450arom Immunoreactivity was observed in adrenal medulla in the three Grs and in subcapsular ZG of Gr 3. Double-immunofluorescence studies revealed that cP450arom and chromogranin A only colocalize in adrenal medulla of subjects younger than 18 months. In these samples, exon 1.b-derived transcript was 3.5-fold higher, while exon 1.a-, 1.c-, and 1.d-derived transcripts were 3.3-, 1.9-, and 1.7-fold lower, respectively, than in subjects older than 6 yr.Conclusions: Our results suggest that estrogens produced locally in adrenal medulla would play a role in zona reticularis functional differentiation through ERβ. The cP450arom and GPR30 expression in subcapsular ZG, colocalizing with a high-cell proliferation index, previously reported, suggests a local GPR30-dependent estrogen action in proliferation and migration of progenitor adrenal cells.

The expression of aromatase, estrogen receptor alpha (ERalpha) and beta (ERbeta), androgen receptor (AR), and cytochrome P-450 side chain cleavage enzyme (cP450scc) was studied in prepubertal testis. Samples were divided in three age groups (GRs): GR1, newborns (1- to 21-d-old neonates, n = 5); GR2, postnatal activation stage (1- to 7-mo-old infants, n = 6); GR3, childhood (12- to 60-mo-old boys, n = 4). Absent or very poor detection of ERalpha by immunohistochemistry in all cells and by mRNA expression was observed. Leydig cells (LCs) of GR1 and GR2 showed strong immunostaining of aromatase and cP450scc but weak staining of ERbeta and AR. Interstitial cells (ICs) and Sertoli cells (SCs) expressed ERbeta, particularly in GR1 and GR2. Strong expression of AR was found in peritubular cells (PCs). For all markers, expression in GR3 was the weakest. In germ cells (GCs), i.e. gonocytes and spermatogonia, aromatase and ERbeta were immunoexpressed strongly whereas no expression of ERalpha, AR, or cP450scc was detected. It is proposed that in newborn and infantile testis, testosterone acting on PCs might modulate infant LC differentiation, whereas the absence of AR in SCs prevents development of spermatogenesis. The role of estrogen is less clear, but it could modulate the preservation of an adequate pool of precursor LCs and GCs.

IGF-1, IGF-2, and type 1 IGF receptor (IGF-R1) mRNA expression and immunolocalization and cell proliferation index were studied in human adrenals from early infancy to late puberty. Adrenals were obtained from transplantation donors or from necropsies of endocrinologically normal subjects. Subjects were divided into three age groups: group 1

Context: 3βHSD2 is a bifunctional microsomal NAD+-dependent enzyme crucial for adrenal and gonad steroid biosynthesis, converting Δ5-steroids to Δ4-steroids. 3βHSD2 deficiency is a rare cause of congenital adrenal hyperplasia caused by recessive loss-of-function HSD3B2 mutations. Objective: The aim was to define the pathogenic consequences of a novel missense mutation in the HSD3B2 gene. Patient: We report a 7-month-old 46,XX girl referred because of precocious pubarche and postnatal clitoromegaly. Hormonal profile showed inadequate glucocorticoid levels, increased 17OHP and renin levels, and very high DHEAS levels, suggestive of compensated nonsalt-losing 3βHSD2 deficiency. Design and Results: Direct sequencing revealed a novel, homozygous, pG250V HSD3B2 mutation. In vitro analysis in intact COS-7 cells showed impaired enzymatic activity for the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione (20% and 27% of WT at 6 h, respectively). G250V-3βHSD2 decreased the Vmax for progesterone synthesis without affecting the Km for pregnenolone. Western blot and immunofluorescence suggested that p.G250V mutation has no effect on the expression and intracellular localization of the mutant protein. Molecular homology modeling predicted that mutant V250 affected an L239-Q251 loop next to a β-sheet structure in the NAD+-binding domain. Conclusions: We identified a novel p.G250V mutation of HSD3B2 which causes an incomplete loss of enzymatic activity, explaining the compensated nonsalt loss phenotype. In vitro and in silico experiments provided insight into the structure-function relationship of the 3βHSD2 protein suggesting the importance of the L239-Q251 loop for the catalytic activity of the otherwise stable 3βHSD2 enzyme. Fil: Baquedano, María Sonia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ciaccio, Marta. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Marino, Roxana Marcela. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Perez Garrido, Natalia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Ramirez, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Maceiras, Mercedes. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina Fil: Turjanski, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Defelipe, Lucas Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Rivarola, Marco Aurelio. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Belgorosky, Alicia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

StAR facilitates cholesterol entry into the mitochondria as part of the transduceosome complex. Recessive mutations in the gen STAR cause classic and nonclassic congenital lipoid adrenal hyperplasia. The aim of the study was to analyze the molecular consequences of a novel heterozygous STAR mutation in a 46,XY patient with ambiguous genitalia and adrenal insufficiency. We found a de novo heterozygous IVS-2AG STAR mutation and the reported heterozygous p.G146A SF1 polymorphism with normal CYP11A1, FDXR, FDX1, VDAC1 and TSPO genes. RT-PCR and sequencing from patient's testicular RNA showed a -exon2 transcript and the wild-type (WT) transcript. Both 37 kDa precursor and 30 kDa mature protein were detected in COS-7 cell transfected with mutant and WT plasmids. Immunofluorescence showed almost no co-localization of mitochondria and mutant protein (delta22-59StAR). Delta22-59StAR activity was 65±13% of WT. Cotransfection with WT and delta22-59StAR plasmids reduced WT activity by 62.0% ± 13.9. Novel splice-junction heterozygous STAR mutation (IVS-2AG) resulted in the in-frame loss of amino acids 22 to 59 in the N-terminal mitochondrial targeting signal. A misfolded p.G22_L59delStAR might interfere with WT StAR activity by blocking the transduceosome complex, causing an autosomal dominant form of StAR deficiency, explaining the clinical phenotype.

Adrenarche is the direct consequence of the organogenesis of the zona reticularis (ZR). Proliferation of cortical cells could take place in the outermost layers of the adrenal cortex. Cells could then migrate to differentiate the zona glomerulosa (ZG) and zona fasciculata (ZF) during fetal life, and the ZR during postnatal life. After adrenarche, there are detectable increases in circulating DHEA and DHEA-S. Adrenarche could result from an increase in 17,20-lyase activity of P450c17 secondary to high levels of cytochrome b5 expression, and from a decrease in 3βHSD2 expression along with an increase in the expression of SULT2A1 in the ZR. The GH-IGF system and insulin, among other factors, might also modulate adrenal androgen production. Furthermore, high concentrations of estradiol enhance basal and ACTH-stimulated DHEA-S production, while aromatase expression was observed in the human adrenal medulla but not in the ZR, suggesting that estrogens produced in the adrenal medulla might be involved in the regulation of androgen production in the ZR. Premature adrenarche might be associated with ovarian hyperandrogenism and polycystic ovarian syndrome in females, as well as with insulin resistance in both sexes. However, many questions remain, transforming adrenal androgens into markers of diseases important for human health.

P450 aromatase (P450Aro), involved in androgen to estrogen conversion, is encoded by the CYP19 gene. P450Aro c655GA mutation described in heterozygous form in a girl and in homozygous form in an adult male with P450Aro deficiency results in an aberrant splicing due to disruption of a donor splice site. A truncated inactive protein would be expected if intron5 is retained. Surprisingly, the girl described with this mutation showed spontaneous breast development and pubertal estradiol (E2) levels suggesting residual P450Aro activity (AA). Formerly, we postulate the in frame E5 skipping as a consequence of this mutation generating a protein with some degree of activity. When P450Aro mRNA expression was analysed from patient's lymphocytes, an aberrant spliced mRNA lacking E5 (-E5mRNA) was detected, suggesting an association between E5 skipping and the presence of the mutation. Splicing assays in Y1 cells confirmed this association. -Ex5 cDNA expression in Y1 cells resulted in an inactive protein that could not explain patient's phenotype. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to splicing mutations and physiological alternative splicing (AS) events. Therefore, -Ex5mRNA was assessed as a natural occurring alternative transcript in normal human steroidogenic tissues. As P450Aro -E5mRNA expression was detected in human term placenta, prepubertal testis and prepubertal adrenal, we might speculate that AS of P450Aro coding region would occur in humans and would be involved in the complex AA regulation. Furthermore, tissue specific regulation of AS might suggest low expression of +E5mRNA from the c655GA allele explaining residual AA evidenced in the affected girl.

OBJECTIVE: The previously described c655G>A mutation of the human cytochrome P450 aromatase gene (P450aro, CYP19) results in aberrant splicing due to disruption of a donor splice site. To explain the phenotype of partial aromatase deficiency observed in a female patient described with this mutation, molecular consequences of the c655G>A mutation were investigated. DESIGN: To investigate whether the c655G>A mutation causes an aberrant spliced mRNA lacking exon 5 (-Ex5), P450aro RNA was analysed from the patient's lymphocytes by reverse transcription polymerase chain reaction (RT-PCR) and by splicing assays performed in Y1 cells transfected with a P450aro -Ex5 expression vector. Aromatase activity of the c655G>A mutant was predicted by three dimensional (3D) protein modelling studies and analysed in transiently transfected Y1 cells. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to both splicing mutations and physiological alternative splicing events. Therefore, expression of the -Ex5 mRNA was also assessed as a possibly naturally occurring alternative splicing transcript in normal human steroidogenic tissues. PATIENTS: An aromatase deficient girl was born with ambiguous genitalia. Elevated serum LH, FSH and androgens, as well as cystic ovaries, were found during prepuberty. At the age of 8.4 years, spontaneous breast development and a 194.6 pmol/l serum oestradiol level was observed. RESULTS: The -Ex5 mRNA was found in lymphocytes of the P450aro deficient girl and her father, who was a carrier of the mutation. Mutant minigene expression resulted in complete exon 5 skipping. As expected from 3D protein modelling, -Ex5 cDNA expression in Y1 cells resulted in loss of P450aro activity. In addition, the -Ex5 mRNA was present in placenta, prepubertal testis and adrenal tissues. CONCLUSIONS: Alternative splicing of exon 5 of the CYP19 gene occurs in the wild type (WT) as well as in the c655G>A mutant. We speculate that for the WT it might function as a regulatory mechanism for aromatization, whereas for the mutant a relative prevalence of the shorter over the full-length protein might explain the phenotype of partial aromatase deficiency.

e13543 Background: The polymorphic CYP2D6 gene shows a high degree of interindividual and interethnic variability that influences its expression and function. CYP2D6 polymorphism influences the met...